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Development of a loop-mediated isothermal amplification assay for rapid detection of Burkholderia mallei
S Mirzai1, S Safi2, N Mossavari3
1Islamic Azad University Graduate of Veterinary Medicine, Faculty of Specialized Veterinary Sciences, Science and Research Branch Tehran Iran.
Abstract:
The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions were found to be at 63ºC for 60 min. The assay showed high specificity and sensitivity. It was concluded that the established LAMP assay is a rapid, sensitive and practical tool for detection of B. mallei and early diagnosis of glanders.
Insights
A new Loop-mediated isothermal amplification (LAMP) technique rapidly detects B. mallei, the cause of glanders in horses. This sensitive assay enables early diagnosis of this contagious equine disease.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Equine Infectious Diseases
Background:
- Glanders, caused by Burkholderia mallei, is a highly contagious and often fatal equine disease.
- Rapid and accurate diagnostic methods are crucial for controlling glanders outbreaks.
- Current diagnostic techniques may lack the speed or sensitivity required for timely intervention.
Purpose of the Study:
- To develop and validate a Loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Burkholderia mallei.
- To establish optimized reaction conditions for the LAMP assay, focusing on temperature and time.
- To evaluate the specificity and sensitivity of the developed LAMP assay against related bacterial species.
Main Methods:
- Design of six specific primers targeting the integrase gene cluster of B. mallei.
- Optimization of LAMP reaction parameters, including temperature (63°C) and incubation time (60 minutes).
- Specificity testing using DNA from B. pseudomallei and Pseudomonas aeruginosa; analysis of LAMP products via visual inspection and UV-visible electrophoresis.
Main Results:
- The optimized LAMP assay successfully detected B. mallei under specific conditions (63°C for 60 min).
- The assay demonstrated high specificity, distinguishing B. mallei from B. pseudomallei and P. aeruginosa.
- Visual and UV-light analysis confirmed the presence of amplification products, indicating high sensitivity.
Conclusions:
- The developed LAMP assay provides a rapid, sensitive, and practical method for B. mallei detection.
- This technique facilitates the early diagnosis of glanders in equines.
- The LAMP assay holds significant potential for routine veterinary diagnostics and disease surveillance.
